Enzymatic proteins are remarkable natural catalysts that selectively catalyze many reactions under relatively mild reaction conditions. Enzymes also offer the potential to perform sterio- and regio-selective reactions not readily accomplished with conventional chemistry. As used herein, the term “enzyme” refers generally to proteins that catalyze biochemical reactions. These “biopolymers” include amide-linked amino acids and typically have molecular weights of 5,000 or greater. A compound for which a particular enzyme catalyzes a reaction is typically referred to as a “substrate” of the enzyme.
In general, six classes or types of enzymes (as classified by the type of reaction that is catalyzed) are recognized. Enzymes catalyzing reduction/oxidation or redox reactions are referred to generally as EC 1 (Enzyme Class 1) Oxidoreductases. Enzymes catalyzing the transfer of specific radicals or groups are referred to generally as EC 2 Transferases. Enzymes catalyzing hydrolysis are referred to generally as EC 3 hydrolases. Enzymes catalyzing removal from or addition to a substrate of specific chemical groups are referred to generally as EC 4 Lyases. Enzymes catalyzing isomeration are referred to generally as EC 5 Isomerases. Enzymes catalyzing combination or binding together of substrate units are referred to generally as EC 6 Ligases.
Enzymes have been known since the early 1960's to be useful tools for detecting the presence of chemical species. Rogers, K. R., Biosensors Bioelectronics, 10, 533 (1995). Generally all enzymatic biosensors function by one of two methods. The enzyme either converts an undetectable compound of interest into another or series of compounds which can be detected with a chemical-based sensor or the enzyme is inhibited by the presence of the compound of interest and the enzyme inhibition is linked to a measurable quantity.
Enzymatic biosensors have been designed to detect a variety of different compounds such as glucose, creatinine, urea, and cholinesterase inhibitors. Parente, A. H., Marques, E. T. Jr., Appl. Biochem. Biotechnol. 37, 3, 267 (1992); Yang, S., Atanasov, P., Wilkins, E., Ann. Biomed. Eng., 23, 6, 833 (1995). U.S. Pat. No. 5,858,186 describes a urea-based biosensor in which substrate hydrolysis is monitored with a pH electrode. U.S. Pat. Nos. 5,945,343 and 5,958,786 describe enzyme-based sensors in which a fluorophere is immobilized in a first polymer layer and an enzyme is separately immobilized in a second polymer layer. The fluorophere layer fluoresces in the presence of ammonia, which is enzymatically produced from urea and creatinine (respectively, with respect to U.S. Pat. Nos. 5,945,343 and 5,958,786). In addition, U.S. Pat. No. 4,324,858 describes the immobilization of cholinesterase within a porous, dry material for the colormetric detection of organophosphorus pesticides and nerve agents.
It is very desirable to develop a device that employs enzyme-based sensors in a continuous manner for monitoring the environment for the presence of target analytes such as pollutants, target industrial chemicals, or other hazardous chemicals. The development of monitoring devices for sampling and for chemical identification and detection has also been previously put to practice.
Much of the art related to device development focuses on equipment for use in laboratories as automated samplers or fluid handling equipment. U.S. Pat. Nos. 4,224,033 and 4,338,280 each describe fluid handling devices that facilitate the hands-free processing of individual liquid samples in a preparatory fashion for later analysis and evaluation. Similarly, U.S. Pat. No. 4,066,412 discloses a device that can carry disposable reagents to aid in monitoring the physical properties of a reaction mixture by passing light through a fixed solution path length.
Other relevant art describes devices that employ specialized components to facilitate the use of particular sensing chemistries and protocols for fluid analysis. U.S. Pat. No. 4,826,759 describes a fluid sampling device that carries two adsorbent layers that are used to bring fluid components into the device and transfer such elements to a second layer for chemical analysis. Others, U.S. Pat. Nos. 4,726,929 and 4,958,295, describe modular devices that handle and analyze fluids in unique ways including disposable sample collection modules and internal vacuum drives, respectively.
The present invention describes an enzyme-based device for the continuous monitoring of gases or liquids for the presence of target enzyme inhibitors. The use of enzymes within analytical devices is not, in itself, novel. U.S. Pat. No. 4,525,704 describes the use of cholinesterases and electrical currents in detecting toxic gases. Other patents describe devices that can be used to detect the presence of enzyme substrates within a specified sample. U.S. Pat. No. 5,223,224 describes an arrangement for flow injection analysis in which sample gases are kept isolated from the environment within the device. U.S. Pat. Nos. 5,504,006 and 5,994,091 both describe sensor devices to sample gas and liquid streams, respectively, for enzyme substrates by linking enzyme activity brought on by the presence of such substrate to a colorimetric signal.